Larval structure of Passalus gravelyi and sexual dimorphism in Passalid larvae

The adults and larvae of Passalidae are subsocial insects commonly found in tropical forests, living in decaying wood gallery systems constructed by adults. Currently, few repots on the larvae of Neotropical Passalidae have been published and information is scarce. In this study, the Passalus (Pertinax) gravelyi Moreira, 1922 larvae is described for the first time, based on ten larval specimens 1 (1° instar), 4 (2o instar), and 5 (3o instar) associated with three adults collected from a single colony at the Parque Nacional do Itatiaia (Itatiaia, Rio de Janeiro, Brazil). The description was carried out based on electronic and digital photographs of diagnostic structures, with some details on the systematic of the species. The larvae of Passalus gravelyi has the general setal ‘Pertinax’ pattern and differed from others by 16 to 18 setae on the anal ring, the other larvae data from Brazilian species show the anal ring with 10 to 12 setae. A discussion on the presence of sexual dimorphism in 62 species of two and three instars of Passalidae larvae is provided for the first time. Besides, a description of the terminal ampulla present as a cuticular structure found in the medial-ventral area of the 9th abdominal sternite in males is also given. The terminal ampulla was only observed in the Passalidae male larvae and was not visible in female larvae. The terminal ampulla are acknowledged now in males of 64 passalid species, that are taxonomically distributed in world tropical forests, at the Oriental and Australian subfamily Aulacocyclinae (Aulacocyclini & Ceracupini) and the cosmotropical subfamily Passalinae (Solenocyclini, Macrolinini, Passalini, & Proculini). Rev. Biol. Trop. 63 (3): 695-704. Epub 2015 September 01.

Larvae of the family Passalidae occur with relative abundance in tropical forest and they are associated with adults in fallen trunks.Only a few studies on the larvae of Neotropical Passalidae have been published (Costa & Fonseca, 1986;Costa, Vanin, & Casari-Chen, 1988;Schuster & Reyes-Castillo, 1981, 1990;Schuster, 1992), and up to date, the most important work on Brazilian species (Costa & Fonseca, 1986) has described the larvae of 21 species.
All known Passalidae are subsocial, with parental adults, immature adults and larvae, all living in a family group in a gallery system constructed by the adults, almost exclusively within or beneath decaying wood (trunks, limbs, roots, and wood particles).This is associated with interesting behaviors including cooperation, parental care, defense, hierarchic systems and communication, including a complex acoustical repertoire (Reyes-Castillo, 1970;Schuster, 1983;Reyes-Castillo & Halffter, 1984).
The subgenus Pertinax in Passalus comprises 40 species, of which 23 have been recorded in Brazil (Hincks & Dibb, 1935;Fonseca & Reyes-Castillo, 2004).Passalus (Pertinax) gravelyi Moreira, 1922 is an endemic species from the Atlantic Forest (Mata Atlântica) and known only from the states of Rio de Janeiro and Minas Gerais, and from elevations above 1 000 m.
Our current research includes a systematic study of the larvae of Neotropical Passalidae.While preparing the description of the 3 rd instar larva of Passalus (Pertinax) gravelyi Moreira, 1922, we noted the occurrence of a sexual dimorphism in the 2 nd and 3rd instar larvae of this species; the male larvae has a terminal ampulla on the margin of the 9th sternite, and investigated the occurrence of this character in the species from all of supra-generic taxa of Passalidae.
The terminal ampulla was first described by Herold (1815) in the larvae of Lepidoptera and defined more specifically by Snodgrass (1941) in Hymenoptera.In Coleoptera the terminal ampulla was described in Scarabaeidae by Hurpin (1953), and subsequently the presence of sexual dimorphism in Passalidae, Lucanidae, Scarabaeidae and Melolonthidae larvae has been reported in literature of Scarabaeoidea (Menees, 1957;Onore, 1994;Martínez & Lumaret, 2003, 2005;Ramírez-Salinas, Pacheco-Flores, Castro-Ramírez, & Morón, 2010).The sexual dimorphism in Passalidae has never been studied in detail, especially in larvae and pupae.The goal of our study was to describe the larvae of Passalus (Pertinax) gravelyi Moreira, 1922, and to begin the systematic study of larvae with the knowledge about the sexual dimorphism in larvae of the Neotropical Passalidae.

MATERIAL AND METHODS
The samples were collected in decayed wood in the Parque Nacional do Itatiaia (Itatiaia, Rio de Janeiro, Brazil); a total of 10 specimens of larvae were collected from the same tunnel system as three adults, and all were preserved in ethanol 96 % until their evaluation.
At least 300 specimens of 62 species (Table 1) were comparatively studied about the terminal ampulla in the following Institutions: Coleção José Alfredo Pinheiro Dutra, Departamento de Zoologia, Universidade Federal do  The morphological characters and the illustrations were made with the aid of a Leica MZ 7.5 stereomicroscope with a drawing tube and integrated digital camera Leica 3.0 CMOS.The larvae terminology followed the primary setal patterns proposed by Schuster & Reyes-Castillo (1981), the general terms of structures as Costa et al. (1988), and the general taxonomic classification followed Boucher (2006).
The electronic photographs were made after the dehydration of some larval structures in a graded ethanol series (70-100 %), air dried, and mounted on aluminum stubs and palladium gold covered.Subsequently, the samples were photographed in a JEOL model JSM 5600 LV Scanning electron Microscope.
In order to study the terminal ampulla, the larvae were fixed following the techniques described by Martínez (1999Martínez ( , 2002) ) using Black of chlorazol and Feulgen-woven green.We investigated the occurrence of the sexual dimorphism in passalid larvae, with the dimorphism present in the structure of the terminal ampulla.We observed the terminal ampulla in male larvae of the 62 passalid species across the two subfamilies and six tribes.

AN OVERVIEW ON SEXUAL DIMORPHISM IN PASSALID LARVAE
In this study sexual dimorphism in passalid larvae is characterized in detail for the first time, with the dimorphism present in the structure of the terminal ampulla (sometimes referred to as "genital ampula").The terminal ampulla has been reported and described in various holometabolous insects (Herold, 1815;Snodgrass, 1941;Hurpin, 1953;Menees, 1957;Matsuda, 1976;Onore, 1994;Martínez & Lumaret, 2005) as the cuticular structure in medial-ventral area of the 9 th abdominal sternite in males (Fig. 10,Fig. 11,Fig. 12,Fig. 13,Fig. 14 and Fig. 15).Ramírez-Salinas et al. (2010) reported that the terminal ampulla (referred to as "genital ámpula") was present in the last (tenth) abdominal sternite of the pupae of the dynastine scarab, Ligyrus nasutus (Burmeister) but that it was less prominent than the terminal ampula in male pupae.In other Scarabaeoidea, the terminal ampulla is evident in all larval instars and it is observed at apex of the aedeagus at the last abdominal ventrite in male pupae (Martínez & Lumaret, 2005).We were unable to detect the terminal ampulla in the first instar passalid larvae, but in the second and third instar larvae and in pupae, the sex of the immatures can be determined.We observed the terminal ampulla only in male larvae of the 62 passalid species across the two subfamilies and six tribes (sensu Boucher, 2006) of Passalidae (Table 1), using the techniques of Martínez (1999) for dissecting and staining the terminal ampulla in the Scarabaeinae.
The terminal ampulla of late-instar passalid male larvae is visible ventrally as a distinct patch of cuticular tissue of the 9th sternite, between and internal to the pair of ventral abdominal setae (AV) of this segment (Fig. 10,Fig. 11,Fig. 12,Fig. 13,Fig. 14 and Fig. 15).This structure, terminal ampulla, it is a very thin layer, with no apparent differences among species.
The occurrence of the sexual dimorphism in passalid larvae was characterized in detail for the first time based in the structure of this terminal ampulla.This structure was localized on the margin of the 9th sternite in male larvae, in species of the supra-generic taxa of Passalidae: all subfamilies and tribes, except the Leptaulacini, sensu Boucher (2006).In this study, we were unable to detect the terminal ampulla in first instar passalid larvae, but in second and third instar male larvae, and pupae (only clearly evaginated aedeagus in males), the sex of immatures was determined for species of subfamilies Aulacocyclinae and Passalinae.Data from this study included the largest world range of information to Passalidae, comprising 62 passalid species, 29 genera in six tribes -Aulacocyclini, two species from Brunei & Australia, and Ceracupini, two species from Japan & Taiwan; Solenocyclini, two species from Ivory Coast; Macrolinini, one species from Brunei; Passalini, 19 species from Mexico, Guatemala, Colombia, Ecuador, Jamaica, Guadeloupe Lesser Antilles & Brazil; and Proculini, 36 species from United States of America, Mexico, Guatemala, Colombia & Brazil)-.Obviously is easier to determine the sexual dimorphism in larvae than in adults, for which the recent studies pointed specific areas of the adult pubescent body (e. g., species of Paxillus, see Mattos & Mermudes, 2013).Finally, the testicle, deferent ducts and other structures observed in Scarabaeidae by Martínez and Lumaret (2005), were not seen in Passalidae species, possibly because sexual maturation occurs later, which can be further investigated.

ACKNOWLEDGMENTS
We would like to thank Imelda Martínez from Instituto de Ecología (INECOL, México) for help with literature and techniques of dissection and pigmentation, and also to Tiburcio Laez Aponte for help with the Scanning electron Microscope photographs.We are grateful to people who made material available, and Alan Gillogly (Orma J. Smith Museum of Natural History) for the revision of the English and anonymous reviewers for comments.This study was supported by FAPERJ (process, 100.927/2011, 100.030/2014) and partially by the project 169604 from the Consejo Nacional de Ciencia y Tecnología (CONACYT, México).